Inhalation anesthetics are valuable agents in widespread clinical use. However, the cellular and molecular mechanisms by which these compounds elicit clinically important actions, such as loss of consciousness and immobility, are still incompletely understood. Accumulating evidence implicates neuronal membrane ion channels as direct targets for anesthetic effects, with much emphasis historically on GABAA and glycine receptors. Our laboratory has recently demonstrated that anesthetics decrease excitability in somatic motoneurons via modulation of two distinct ion channels: activation of background or 'leak' K+ channels and inhibition of hyperpolarization-activated cationic channels (Ih). This channel modulation occurs at clinically relevant concentrations and the motoneuronal inhibition that results could account, at least in part, for the immobilizing effects of anesthetics. The relatively recent cloning of KCNK and HCN channel families, the substrates for neuronal leak K+ and Ih, channels, provides an opportunity to determine molecular mechanisms underlying anesthetic effects on these channels. Our published and preliminary data indicate that the anesthetic- activated K+ current in motoneurons involves the pH- and neurotransmitter-sensitive TASK-1 (KCNK3) and TASK-3 (KCNK9) channel subunits, either in homo- or heteromeric configurations; anesthetic effects on these leak K+ channels appear to be modulated by neurotransmitter action. Likewise, the cyclic- nucleotide-gated HCN1 and HCN2 subunits are co-expressed in motoneurons, where they also may associate into homo- or heteromeric channels; our preliminary data indicate that volatile anesthetics affect homomeric HCN subunits differentially and that cAMP modulates effects of anesthetic. We hypothesize that the effects of volatile anesthetics on neuronal leak K+ currents and Ih, and their modulation by neurotransmitters, are fully recapitulated in cloned TASK and HCN channels, and that these channels include determinants critical for anesthetic effects within their primary structure. The Specific Aims are: [1] Elucidate molecular mechanisms underlying volatile anesthetic effects on 'leak' K+ (TASK) channels; [2] Elucidate molecular mechanisms underlying volatile anesthetic effects on hyperpolarization-activated cationic (HCN) channels. For these studies, we record cloned TASK and HCN channel currents in a mammalian heterologous expression system and native leak K+ currents and Ih in motoneurons. We characterize anesthetic effects and their modulation by neurotransmitters (or cAMP) on cloned and native channels, and use site-directed mutagenesis in order to identify channel domains that are necessary for these actions. These experiments will determine molecular mechanisms by which volatile anesthetics modulate TASK and HCN channels native to motoneurons, with implications for their immobilizing actions. Widespread expression of these channels in the CNS suggests that their modulation by anesthetics in other brain regions may contribute to additional anesthetic actions.